LexA site

FIGURE 17-4 Domain swap experiment Part (a) shows that the DNA-binding domain of Gall, without that protein's activation domain, can still bind DNA, but cannot activate transaction. In another experiment {not shown) the activation domain, without the DNA binding domain, also does not activate transcription. Part (b) shows that attaching the activation domain of Cai4 to the DNA-binding domain of the bacteria! protein Le,¬ĽA creates a hybrid protein that activates transcription of a gene in yeast as long as that gene bears a binding site lor LexA. Bpression is measured using a it-porter plasmid in which the GAL! promoter is fused to the F, colt lacZ gene whose product (p-gatactosidase) is readily assayed m yeast cells. Levels of expression from the GAL! promoter in response to the various activator constructs can therefore easily be measured. Similar reporter plfsmids are used in many experiments in this chapter.

Box 17-1 The Two Hybrid Assay

This assay is used to identify proteins that interact with each other. Thus, in the case shown in Box 17-1 Figure 1, activation of a reporter gene depends on the fact that protetn A interacts with protein B (even though those proteins need not themselves normally have a role in transcriptional activation) The assay is predicated on the finding, discussed in the text, that the DNA-bindtng domain and activating region can be on separate proteins, as long as those proteins interact, and the activating region is thereby tethered to the DNA near the gene to be activated. Practically, the assay is carried out as follows. The gene encoding protein A is fused to a DNA fragment encoding the DNA-btnding domain of Gal4. The gene fof a second protein (B) is fused to a fragment encoding an activating region. Neither protein alone, when expressed in a yeast cell, activates the reporter gene carrying Gal4 binding Sites (as shown in the first two lines of the figure). When both hybrid genes are expressed together in a yeast cell, however, the interaction between proteins A and B generates a complete activator, and the reporter is expressed, as shown in the bottom line of the figure. In a widely used elaboration of this simple assay, the two hybrid assay is employed to screen a library of candidates to find any protein that will interact with a known starting protein. So now, protein A in the figure would be the starting protein (called the "bait"), while protein B (the "pre/') represents one of many alternatives encoded by the library (see Chapter 20 for a description of how libraries are made). Yeast cells are transferred with the construct encoding protetn A fused to the DNA-binding domain, together with the library encoding many unknown proteins fused to the activating region. Thus, each transfected yeast cell contains protetn A tethered to DNA and one or another alternative protein B fused tu do activating region. Any celt containing a combination of A and B that interacts will activate the reporter gene. 5uch a cell will form a colony that can be identified by plating on suitable indicator medium. Typically the reporter gene would be lacZ, and positive colonies (those comprising cells expressing the reporter gene) would be blue on appropriate indicator plates.

534 Gent Regulation in Eukaryotes Box 17-1 {Continued)

Was this article helpful?

0 0

Post a comment